Pharmacodynamics of fluoroquinolones. A. Dalhoff* Bayer AG, Pharma Research Centre, PO Box , Wuppertal, Germany

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1 Journal of Antimicrobial Chemotherapy (1999) 43, Suppl. B, Pharmacodynamics of fluoroquinolones JAC A. Dalhoff* Bayer AG, Pharma Research Centre, PO Box , Wuppertal, Germany Fluctuating concentrations of three fluoroquinolones (moxifloxacin, sparfloxacin and ofloxacin) and a β-lactam (amoxycillin) were used in vitro to simulate antibiotic concentrations in human serum after oral doses of antibiotics. The antibiotics were tested against Staphylococcus aureus and Streptococcus pneumoniae Moxifloxacin and sparfloxacin were also tested against Escherichia coli Neumann. Human serum concentrations of moxifloxacin and ciprofloxacin were also simulated in an in-vivo murine thigh muscle model against S. aureus, S. pneumoniae and E. coli. Ciprofloxacin, sparfloxacin and ofloxacin had a doseindependent effect on Gram-positive organisms beyond their optimal dose that gave a maximum effect, as did amoxycillin. In contrast, moxifloxacin had a dose-dependent and therefore concentration-dependent effect on both Gram-positive and β-lactam-susceptible and-resistant Gram-negative organisms. The marked activity of moxifloxacin against both Gram-positive and Gram-negative organisms was confirmed in an in-vivo model. A human dose equivalent of 200 mg moxifloxacin reduced viable counts of S. pneumoniae below the limit of detection and regrowth did not occur. S. aureus was eliminated almost as effectively as S. pneumoniae. A 200 mg dose of moxifloxacin completely eliminated the original inoculum of E. coli within 6 h. Treatment of S. aureus with ciprofloxacin (250 or 500 mg) resulted in a dose-independent decrease in viable counts by approximately 3.5 log 10 cfu/ml. A 125 mg dose of ciprofloxacin almost completely eliminated the original inoculum of E. coli within 8 h, whereas both the 250 mg and 500 mg doses reduced viable counts below the limit of detection. Thus, the in-vitro and in-vivo pharmacodynamic models used in this study established that moxifloxacin was highly effective against both Gram-negative and Gram-positive bacteria. Introduction Many methods have been used to evaluate in-vitro activities of antibacterial agents. Most commonly, MICs are determined; less frequently, MBCs are also measured. MICs and MBCs are assessed by exposing bacteria to a constant antibiotic concentration for approximately 18 h. Such static measures provide information on inhibition or killing at an endpoint of 18 h incubation. However, these endpoint measurements are, by nature, discrete and do not provide information on the time-dependent killing rate during the incubation period. 1 3 This can be achieved by performing time kill experiments that use constant antibiotic concentrations. 4 However, antibiotic concentrations in vivo fluctuate according to the pharmacokinetic properties of the drug. 5 7 Therefore models that interpret both pharmacokinetics and bacterial response may provide more clinically meaningful information about the potential efficacy of an antibiotic. First-generation fluoroquinolones (e.g. ciprofloxacin and ofloxacin) are highly active against aerobic or facultative Gram-negative bacilli These fluoroquinolones have concentration-dependent killing rates for Gramnegative organisms; in this, they resemble aminoglycosides rather than β-lactams. 1,3 Therefore, it would be of interest to confirm the results achieved with constant antibiotic concentrations, when the fluctuating antibiotic concentrations in human serum are simulated. First-generation fluoroquinolones are not as active against Gram-positive bacteria as they are against Gram-negative bacteria. Newer fluoroquinolones, such as moxifloxacin, are characterized by enhanced activity against Gram-positive bacteria with maintenance of activity against Gram-negative bacteria Moxifloxacin exhibits concentration-dependent activity against both Gram-positive and Gram-negative bacteria In-vitro modelling has helped describe the dose response relationship of fluoroquinolones, especially *Tel: ; Fax: ; Axel.Dalhoff.AD@Bayer-ag.de 1999 The British Society for Antimicrobial Chemotherapy 51

2 A. Dalhoff ciprofloxacin. In-vitro simulation of human serum antibiotic concentrations revealed that test organisms were killed in a concentration-dependent manner. 13,21,23 27 Various in-vivo models of infection corroborated the predictive validity of in-vitro models Some details of the antibacterial efficacy of moxifloxacin concentrations simulating human serum kinetics have been described previously. 5 This report provides more comprehensive information. Heidelberg, Germany) which was fixed within the central compartment. Antibiotics selectively diffused into the tubing whereas bacteria could not penetrate into the rest of the central compartment. At the same time points as quantification of colonyforming units (cfu), the antibiotic concentrations were measured by a conventional cup-plate agar diffusion test with Bacillus subtilis spore suspension as the indicator organism. 33 Materials and methods Bacterial strains The following bacteria were used: Escherichia coli Neumann and Staphylococcus aureus 12241, originating from the strain collection of Bayer AG; Streptococcus pneumo - niae 4241, provided by C. Carbon (Paris, France); S. pneu - moniae 8972 (penicillin-sensitive, MIC 0.06 mg/l); S. pneumoniae 8973 (intermediately sensitive to penicillin, MIC 0.25 mg/l); and S. pneumoniae 8974 (penicillinresistant, MIC 4 mg/l). The latter three strains were obtained from B. Wiedemann (Bonn, Germany) and are recent clinical isolates. Antibiotics The following fluoroquinolone antibiotics were used: moxifloxacin and ciprofloxacin (Bayer AG, Wuppertal, Germany); sparfloxacin (Rhône Poulenc Rorer, Cologne, Germany); and ofloxacin (Hoechst Marion Roussell, Frankfurt, Germany). Amoxycillin (SmithKline Beecham, Munich, Germany) was also used. In-vitro model A slightly modified one-compartment in-vitro model 32 was used. Briefly, this model consists of a central compartment into which the antibiotics to be studied are pumped via programmable pumps until the maximum serum concentration to be simulated is reached. Thereafter, antibioticfree medium is pumped into the central compartment and is continuously eliminated in parallel to mimic t ½ values. Control growth in the absence of antibiotics was studied in the same model. Brain heart infusion (BHI) broth (Oxoid, Wesel, Germany) was used for growing Gram-negative and Grampositive bacteria. S. pneumoniae was incubated in BHI supplemented with 5% bovine serum. Viable counts were determined at 0, 0.5, 1, 2, 2.5, 3, 4, 6, 8, 12 and 24 h. All antibiotics were only dosed once. In order to avoid a wash-out of bacteria from the central compartment when simulating serum antibiotic concentration time profiles for antibiotics with short half-lives (e.g. amoxycillin, t ½ 1 h), the bacteria were placed into dialysis tubing (Neolab, In-vivo model The neutropenic mouse thigh model described by Gerber et al. 34 was used to study the effect of antibiotics in vivo. Female CF1 outbred mice (Winkelmann, Borchen, Germany) weighing c. 25 g were used. Induction of neutropenia. Cyclophosphamide was injected intraperitoneally as doses of 150 mg/kg and 100 mg/kg on days 5 and 1, respectively. Cyclophosphamide reduced total leucocyte counts from 12,700/ L on day 0 to 500/ L on day 5 and maintained this low leucocyte count for a further 6 days; this reduction in leucocyte counts was accompanied by a reduction in granulocyte counts. Infection of animals. Half an hour before antibiotic treatment, the mice were infected with 0.05 ml of a suspension containing 10 5 cfu of E. coli Neumann, S. aureus or S. pneumoniae Bacterial suspensions were injected into the right thigh. Twelve animals were infected per treatment group. Treatment of animals. Two groups of mice were treated with either ciprofloxacin or moxifloxacin. Antibiotics were administered subcutaneously (0.1 ml) every 15 min to simulate human serum concentrations of these two fluoroquinolones. Control mice received physiological saline at the same dosing intervals. Fractionated dosing was stopped after 8 h: thereafter, animals did not receive any antibiotics. Quantification of bacterial growth. At various time intervals (4, 8 and 24 h), three mice per sampling point were killed for quantification of bacteria per thigh. Thighs were removed aseptically and were immediately homogenized in physiological saline (ph 3.0) using a Potter S h o m o- genizer (Braun, Melsungen, Germany). Homogenates were placed on DST agar plates (Oxoid) in triplicate and following incubation of the plates for 18 h, the cfu/ml thigh homogenate were calculated. Bioassay. Thigh muscle concentrations of moxifloxacin and ciprofloxacin were measured using the conventional cup-plate agar diffusion assay, 33 with a B. subtilis spore suspension (Difco Laboratories, Detroit, MI, USA) as indication organism in Isosensitest agar (Difco Laboratories). Holes punched into the agar were filled with 100 L 52

3 of the sample or a standard solution ranging from 2 mg/l to 0.06 mg/l (1:1 dilution). Plates were incubated at 37 C for 18 h. Pharmacodynamics of fluoroquinolones Results In vitro Moxifloxacin. Exposure of S. pneumoniae to fluctuating concentrations of moxifloxacin, simulating human serum concentrations following single oral doses of 100, 200, 400 and 600 mg, resulted in a dose-dependent reduction in viable counts. The inoculum was eliminated within 8 12 h. This effect was independent of the strain of organisms used: S. pneumoniae strain 4241, frequently used in studies of experimental pneumonia, was killed as effectively as the clinical isolates 8972, 8973 and 8979 (Figure 1). Similarly, viable counts of S. aureus were reduced in a dose-dependent manner and the inoculum was also eliminated within 8 12 h incubation by concentrations that simulated doses of 100, 200, 400 and 600 mg in humans. Following a dose of 100 mg, viable counts of S. aureus were reduced less rapidly than with the higher doses, but viable counts were also no longer detectable at the end of the incubation period (Figure 2a). Gram-negative bacteria were affected to a much greater extent than Grampositive organisms. The inoculum of E. coli was eliminated within 2 4 h even with moxifloxacin concentrations simulating a dose of 100 mg (Figure 2b). Similar results were obtained against Gram-negative organisms with all other fluoroquinolones and with amoxycillin (data not shown). Regrowth did not occur with any of the Gram-negative or Gram-positive strains tested. Sparfloxacin. Simulation of human serum concentrations of sparfloxacin following an oral dose of 200 mg resulted in a reduction in viable counts of S. aureus by only 1 log 10 and regrowth occurred after 12 h. Doubling the antibiotic concentration, thus simulating serum concentrations after a 400 mg oral dose, resulted in a 4 log 10 decrease in viable counts within 6 h and prevented regrowth thereafter (Figure 3). S. pneumoniae 4241 viable counts were reduced by 1.5 log 10 titres within 8 12 h by sparfloxacin concentrations simulating serum concentrations after a 200 mg oral dose. Doubling this concentration (400 mg) increased the anti-pneumococcal effect by only 0.5 log 10 titre at 8 12 h (Figure 3). Ofloxacin. Concentrations simulating an oral dose of 200 mg reduced viable counts of S. pneumoniae 4241 by only 1 log 10 titres within 4 h; afterwards, regrowth occurred rapidly and viable counts at 12 h were not significantly different from those of the drug-free control (Figure 4). Doubling the ofloxacin concentration to simulate concentrations after a 400 mg oral dose reduced viable counts of S. pneumoniae by 4 log 10 titres and a moderate regrowth of Figure 1. Antipneumococcal effects of fluctuating moxifloxacin concentrations simulating human serum concentrations following a single oral dose of 100 ( ), 200 ( ), 400 ( ) or 600 ( ) mg, against (a) S. pneumoniae 4241; (b) S. pneumoniae 8973 (penicillin-intermediate-susceptible); (c) S. pneumoniae 8974 (penicillin-resistant), control. Data for S. pneumoniae 8972 (penicillin-sensitive) were virtually identical to those for S. pneumoniae approximately 1 log 10 units subsequently occurred (Figure 4). Viable counts of S. aureus were reduced by 5 log 10 titres within 8 h; upon exposure to concentrations simulating a 200 mg dose: however, regrowth occurred thereafter (Figure 4). Within the first 8 h of exposure, the anti-staphylococcal activity of ofloxacin was not significantly enhanced by doubling the concentration used: viable counts were reduced by only a further 0.4 log 10 titres. However, a significant regrowth by 4 log 10 titres was noted when serum 53

4 A. Dalhoff Figure 2. Efficacy of fluctuating moxifloxacin concentrations following a single oral dose of 100 ( ), 200 ( ), 400 ( ) or 600 ( ) mg, against (a) S. aureus 12241; (b) E. coli (Neumann)., control. Figure 3. Efficacy of sparfloxacin concentrations simulating human serum concentrations following a single oral dose of 200 ( ) or 400 ( ) mg against S. aureus ( ) or S. pneumoniae 4241 ( )., control. Figure 5. Effect of fluctuating amoxycillin concentrations simulating human serum levels following a single oral dose of either 250 mg ( ) or 500 mg ( ) against S. aureus ( ) or S. pneumoniae 4241 ( )., control. concentrations following an oral dose of 200 mg were simulated, whereas simulation of a 400 mg dose permitted regrowth by 2 log 10 titres. Amoxycillin. Concentrations simulating amoxycillin serum concentrations after oral doses of 250 mg and 500 mg reduced viable counts and rapidly eliminated S. pneumo - niae 4241 within 6 h. Viable counts of S. aureus were reduced more slowly by 4.5 log 10 titres within 12 h. The effect on both S. pneumoniae and S. aureus was concentration-independent (Figure 5). Regrowth of S. pneumoniae did not occur whereas regrowth of S. aureus by 2 log 10 titres was observed within 24 h. Figure 4. Effect of fluctuating ofloxacin concentrations simulating human serum levels following a single oral dose of either 200 ( ) or 400 ( ) mg against S. aureus ( ) or S. pneumoniae 4241 ( )., control. In vivo Moxifloxacin. Fractionated dosing of moxifloxacin and ciprofloxacin (Figure 6) resulted in thigh muscle concentra- 54

5 Pharmacodynamics of fluoroquinolones Figure 6. Dosing regimen (sc) of (a) moxifloxacin or (b) ciprofloxacin (indicated by the bars) used to mimic human serum concentrations in murine thigh muscle ( ) in the neutropenic mouse thigh-muscle model. tions that closely resembled those following an oral moxifloxacin dose of 200 mg or an oral ciprofloxacin dose of 250 mg in humans. The mean peak thigh muscle concentration of moxifloxacin was 1.0 mg/kg, which corresponds to a mean maximum serum concentration in humans after an oral 200 mg dose. 35 As moxifloxacin concentrations in the thighs of immunosuppressed animals were linearly dose dependent (data not shown), halving of fractionated antibiotic concentrations resulted in thigh muscle moxifloxacin concentrations simulating those after a 100 mg oral dose in humans. Conversely, doubling moxifloxacin concentrations simulated a human oral dose of 400 mg. Thigh muscle concentrations of the antibiotic decreased slowly (t ½ 14 h). Upon cessation of fractionated dosing, moxifloxacin concentrations decreased with a short halflife typical of that found in small rodents, i.e. 1.3 h (data not shown). Treatment of Gram-positive infections in immunosuppressed animals with moxifloxacin was highly effective (Table I). At 12 h, human dose equivalents of 200 mg and 400 mg reduced viable counts of S. pneumoniae 4241 below the limit of detection and regrowth did not occur. S. aureus was eliminated almost as effectively as S. pneu - moniae 4241, although the original inoculum was not completely eliminated; viable counts were reduced in a dose-dependent manner. A 200 mg dose of moxifloxacin completely eliminated the original inoculum of E. coli Neumann by the first sampling point (4 h) (Table I). At lower doses of 25 mg, 50 mg and 100 mg, the viable counts of E. coli decreased in a dose-dependent manner: at 8 h viable counts ranged from 2.8 to 3.65 log 10 cfu/ml following these lower doses (Table I). A moderate regrowth of all test organisms was seen in Table I. Therapeutic efficacy (log 10 cfu/ml thigh homogenate) of moxifloxacin in the mouse thigh infection model in immunosuppressed mice. Moxifloxacin concentrations in the thigh mimicked human serum concentrations following the doses indicated. Moxifloxacin Species/strain Sampling time (h) 100 mg 200 mg 400 mg Control S. aureus S. pneumoniae E. coli Neumann a 3.65 b 3.12 c a 3.12 b 2.87 c a 3.65 b 3.12 c 9.27 a Dose 25 mg. b Dose 50 mg. c Dose 100 mg. 55

6 A. Dalhoff Table II. Therapeutic efficacy of ciprofloxacin (log 10 cfu/ml thigh homogenate) in the mouse thigh infection model in immunosuppressed mice. Ciprofloxacin concentrations in the thigh resembled/mimicked human serum ciprofloxacin concentrations following the doses indicated. Ciprofloxacin Species/strain Sampling time (h) 250 mg 500 mg Control S. aureus E. coli Neumann a 1.80 b a 1.0 b a 2.54 b 8.87 a Dose 125 mg. b Dose 250 mg. all treatment groups at 24 h. This may be due to the fact that dosing was stopped at 8 h and concentrations at the focus of infection declined rapidly. Ciprofloxacin. Monitoring of thigh muscle concentrations following fractionated doses of ciprofloxacin (Figure 6) produced mean peak concentrations of 1.8 mg/kg of muscle homogenate. A dose reduction of 50% caused a corresponding reduction of thigh muscle ciprofloxacin concentrations of 50% (data not shown). These ciprofloxacin concentrations correspond to human serum concentrations of the antibiotic following oral doses of 250 mg and 500 mg, respectively. Thigh muscle concentrations of ciprofloxacin decreased with a t ½ of 5.2 h, which again corresponds to that seen in humans. Upon cessation of fractionated dosing, ciprofloxacin concentrations decreased with a short halflife typical of that seen in small rodents, i.e. 2.3 h. Treatment of S. aureus infections with ciprofloxacin doses of 250 mg and 500 mg resulted in a doseindependent decrease in viable counts by approximately 3.5 log 10 cfu/ml (Table II). Treatment with doses of 250 mg and 500 mg resulted in a reduction of viable counts by 2.9 and 3.18 log 10 titres, respectively, within 4 h, and by 4.17 and 4.22 log 10 titres, respectively, within 8 h, as compared with the drug-free controls. Against E. coli Neumann, ciprofloxacin was highly effective at reducing viable counts in a dose-dependent fashion (Table II). A 125 mg dose almost completely eliminated the original inoculum of E. coli by 8 h. Following 250 mg or 500 mg (data not shown) doses, viable counts were reduced below the limit of detection. A slight regrowth of S. aureus and E. coli was noted in all treatment groups at 24 h possibly because of the rapid decline of thigh-muscle concentrations immediately after cessation of fractionated dosing. Discussion Numerous in-vitro, in-vivo and clinical studies have evaluated the pharmacodynamics of various antibacterial agents. The efficacy of β-lactams is determined by the time for which serum antibiotic concentrations exceed the MIC for the causative pathogen, whereas the efficacy of aminoglycosides depends on concentration and, thus, on dose. 1,3,37 44 Generally, fluoroquinolones are considered to act in a concentration-dependent manner ,28,44 46 However, as earlier fluoroquinolones, e.g. ofloxacin, exert their most pronounced antibacterial effect against Gramnegative organisms, this hypothesis has been proven preclinically and clinically against Gram-negative species, but not against Gram-positive organisms. Sparfloxacin also exerts a concentration-independent effect against Gram-positive organisms in contrast to its effect against Gram-negative organisms. Beyond the concentration producing a maximal effect, higher fluoroquinolone concentrations do not further augment activity against Grampositive bacteria. 1,20,36,49,50 In agreement with these findings, the results from this study clearly demonstrated that ofloxacin and sparfloxacin reduced viable counts of the Gram-positive organisms investigated independent of the dose used. With the exception of the 200 mg dose of ofloxacin, which was ineffective against S. pneumoniae, the antipneumococcal or antistaphylococcal effects of neither ofloxacin nor sparfloxacin could be enhanced by doubling the simulated serum concentrations. As expected, against Gram-positive organisms, a 250 mg dose of amoxycillin was as effective against both S. aureus and S. pneumoniae as a 500 mg dose. In contrast, moxifloxacin s activity was clearly dosedependent and, therefore, concentration-dependent throughout the broad dose range studied, irrespective of whether Gram-negative or Gram-positive organisms were 56

7 Pharmacodynamics of fluoroquinolones exposed. These data agree very well with previous results For Gram-positive bacteria, this effect was observed with both β-lactam-susceptible and -resistant organisms. This marked activity of moxifloxacin against both Gram-negative and Gram-positive bacteria was confirmed in an in-vivo model. In the neutropenic thigh muscle model simulation of human serum concentrations after a single dose of 200 mg of moxifloxacin eliminated both S. pneumoniaeand E. coli from the focus of infection within 8 h. This is in very good accordance with the results from the in-vitro pharmacodynamic model. Furthermore, in the in-vivo model moxifloxacin decreased the viable counts of S. aureus, S. pneumoniae and E. coli dose- and thus concentration-dependently. In contrast, ciprofloxacin exerted a dose-dependent effect against E. coli only, whereas the viable counts of S. aureus were reduced in a concentration-independent manner. The therapeutic efficacy of ciprofloxacin at these doses was not tested against S. pneumoniae, as it had previously been shown that serum ciprofloxacin concentrations were only marginally effective against this organism. Simulation of lung mucosa concentrations of ciprofloxacin, however, reduced viable counts of S. pneumoniae as effectively as, e.g., sparfloxacin. 36 These results show that moxifloxacin is characterized by a significantly improved activity against Gram-positive bacteria and maintenance of activity against Gramnegative organisms compared with first-generation fluoroquinolones. 18,20,51 53 Furthermore, the concentrationdependent effect of moxifloxacin against Gram-positive bacteria, as opposed to the concentration-independent effect of previous fluoroquinolones, may indicate that moxifloxacin is characterized by a higher intrinsic activity and/or a modified target specificity. Moxifloxacin pharmacodynamics also differed from those of amoxycillin which has a concentration-dependent bactericidal effect up to a point of maximum effect; beyond this point increasing amoxycillin concentrations neither enhance the rate of killing nor the total number of bacteria killed. 1,20,49 Moxifloxacin serum concentrations simulated in this study are likely to underestimate clinically-relevant moxifloxacin concentrations. A geometric mean serum concentration versus time curve after a single dose was mimicked with a C max of 2.5 mg/l; thus, mean peak concentrations represented neither steady-state concentrations (which are c. 30% higher) nor mean C max concentrations, which are 3.5 mg/l. Thus, concentrations simulated in this study after a 200 mg dose, for example, mimicked serum antibiotic concentrations at steady-state following repeated doses of 100 mg. Furthermore, moxifloxacin concentrations at the site of respiratory tract infections are significantly higher than the corresponding serum concentrations since the ratios between bronchial mucosa or epithelial lining fluid and the respective serum concentrations are c. 2 7, throughout the sampling period up to 24 h. Additionally, intraphagocytic moxifloxacin concentrations are times higher than the respective serum concentrations. 54 Despite this, integration of pharmacokinetics and the antibacterial activity of moxifloxacin in these in-vitro and in-vivo pharmacodynamic models established that moxifloxacin was highly effective against both Gram-negative and Gram-positive bacteria, particularly against causative pathogens of respiratory tract, skin and skin-structure infections. References 1. Craig, W. A. & Ebert, S. C. (1991). Killing and regrowth of bacteria in vitro: a review. Scandinavian Journal of Infectious Diseases. Supplementum 74, Rustige, C. & Wiedemann, B. (1991). 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